Over 90% of the human proteins contain cysteines, while in silico digest of the human proteome revealed that only about 15% of all human tryptic peptides detectable by mass spectroscopy (MS) contain at least one cysteine in their sequence. This observation combined with the presence of a highly reactive thiol group on its side chain makes cysteine an attractive target for chemical labeling. Cysteine is the only coded amino acid that carries a nucleophilic sulfhydryl (or thiol) group (—SH), which largely exceeds the reactivity of any other nucleophilic function susceptible to be present in proteins. As a result, chemospecific cysteine derivatization is by far the most widely used method for chemical tagging of proteins. Among the vast number of chemical cysteine modification methods reported in literature so far, reagents such as N-substituted maleimides, 4-vinylpyridines and iodoacetamides are most commonly used. All of them possess drawbacks preventing them from being ideal methodology for cysteine labeling, though being suited for this task. These drawbacks are mainly presence of undesired side reactions, in particular for iodoacetamides and maleimides, and instability of addition product in biological environments due to reversible thiol exchange and other side reactions.
The present invention relates to a process for labeling compounds comprising at least one thiol moiety, such as cysteine, with compounds comprising a tag moiety and a 3-arylpropiolonitrile moiety. Said compounds and their addition products with cysteine derivatives show an unexpected stability in a wide range of conditions. The process for labeling compounds comprising thiol moieties of the invention can thus be used for a wide range of applications.